Hi,
I have been using thermo T4 ligase to do ligation for pNL43 constructs. I followed the protocol of life tech to do chemical transformation into STBL2. I used Genehog (DH10B derivative) as transformation control which gives me around 10^5 cfu/ug. STBL2 is
Hi Nanxi,
I assume that you are speaking of plasmid pNL4-3, not 43 as such, right?
The pNL4-3 vector is a huge one (close to15 kb). That means it will be not easy at all to use in ANY transformation. From what I read, with a positive control you get 10^5 cfu/µg. That is for some strange plasmid, what would be your figure with pUC18 or pUC19 (which are the regular control plasmids)?
Typically, for large vectors, such as pNL4-3, you need supercompetent cells (in the range of 10^8 to 10^9 cfu/µg with a pUC plasmid) to get a few transformants per Petri dish !!!!Under 10^8 cfu/µg, we usually get NOTHING, as you did.
My advice is that you buy supercompetent cells from any manufacturer!
Best regards,
Pr Philippe Urban
Hi there,
What is clear from your numbers is that STBL2 is much less competent than Genehog. This is the issue.
An other thing to consider is that obviously you are transforming the recipient strain in order to isolate a specific construct from a ligation mix. If the goal of the ligation is not a library construction, you don't actually need a lot of transformants to get the right construct. So from a ligation mix containing 100ng of plasmid, tens of transformants should be enough for successful screening. If you run in parallel the control experiments (open vector alone without ligase, open vector with ligase), it will give you an idea about what could be the clones obtained from your ligation. Ideally there shouldn't be any clones on controls and then clones from the full ligation are highly proned to be the one you are looking for.
And finally plate the totality of your transformation in order to increase the number of recovered clones.
HI Phillippe,thank you! I used STBL2 which is claimed to have 10^8-10^9 cfu/ug (probably only for small plasmid?). I will try pUC18 to see what happens.
Hi Dominique, thanks a lot! I understand I don't have to have that many transformants on a plate. But normally I get less than 10 for vector+insert. A lot of times I got only 1-2 colonies and after digestion they might not be correct. For vector only, I got nothing. It's recommended to use STBL2 to reduce recombination of plasmids, but it's always not as efficient as it stated. Genehog does give much higher chance of recombination according to digestion. So I am getting confused with pNL43 cloning. Does this happen a lot or it's because of my technique?
Hi,
Transformation of pNL4.3 is so hard because of it's large structure. According to my experience, if you hold competent cells on ice (re-suspended in calcium chloride 100 mM) for 18 hrs and then transform into these competent cells you will get better results.
Hi Nanxi,
I think the issue here is not transformation but ligation efficiency. Cloning in pNL4-3 is not as efficient as in smaller vectors. You could add a transformation control with uncut pNL4-3 to make sure your transformation technique is adequate. Here is a protocol that I used to successfully cloned several ~2kb fragments in pNL4-3 using the Takara Long ligation kit. For every ligation reaction, use 150ng of linearized vector, and a 3-fold molar excess of insert (i.e. 100ng for a 2kb insert) in a 50µl reaction. Add 1µl of ligase and incubate overnight at 16°C. You can skip the 65°C step prior to add the ligase in the ligation protocol. The next day, transform 15µl of the ligation mix into 50µl of XL10 ultracompetent cells and plate the entire transformation. With pNL4-3 it is best to perform all the incubation (including recovery) at 30°C to avoid recombinations. The colonies will take more than 24h to grow. You could start with your current ligase and competent cells first to see if longer ligation with increased DNA amounts will suffice to solve your problem.
Hi Vincent,
Thanks a lot for your advice. Do you purify the ligation mix prior to mix with XL10 ultraconpetent cells? Do you ever have random mutations or recombination issue with XL10 cells?
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